Archive for January, 2009

On Feb 3rd in our previous blog entry on fluorescent proteins, we discussed some basic tips on setting yourself up for success with fluorescent protein based experiments. Here are some more ideas to help boost your imaging success:

1. Know your background.

All cells contain endogenous fluorescent materials which can confound image interpretation, especially when your fluorescent protein signal is weak. Make sure you’re familiar with the autofluorescence of your cell type before starting your FP experiments: take some images of non- expressing control cells using the same filters and excitation wavelength as you plan to use for your FP imaging.

Keep in mind that for mammalian cells, autofluorescence is confined mainly to the blue and green regions of the visual spectrum, while in other organisms (e.g. plants, yeast, and bacteria), some cell types may contain fluorescent compounds in other regions of the spectrum. For any given species and cell type, there is likely to be a wavelength “window” with the least autofluorescence; try to choose a fluorescent protein in this wavelength range for maximum signal above background.

2. Sometimes two (or more) FPs are better than one.

If you are having trouble obtaining sufficient fluorescent signal from a fluorescent protein fusion construct, consider adding an extra copy of the fluorescent protein to boost your brightness. While this is not recommended unless all else has failed, for low-abundance proteins it can substantially increase the likelihood of detection. It is possible to create a functional fusion of two or more copies of fluorescent protein in many cases, although the larger size of such a tag increases the chances of mislocalization, so proper controls and validation are essential if you use this technique. Also, remember that it is generally difficult to use PCR to amplify tandem copies of any gene, including FPs, so restriction-based subcloning is the most reliable way to create multi-copy FP tags.

3. The best fluorescent proteins don’t stick together!

Truly monomeric fluorescent proteins make the best fusion tags, since they don’t produce localization artifacts due to multimerization. Even weak dimers, such as EGFP and its derivatives, can cause trouble if your fusion protein is at high concentration or in a confined space like a membrane or vesicle.

Are you still using your old EGFP fusion constructs? If so, make sure to validate your localization results by other methods, or switch to a truly monomeric FP such as mTFP1 or mWasabi. If you prefer to keep your original constructs, note that any Aequorea GFP-derived FP can be made completely monomeric by adding the A206K mutation.

One final warning — many commercially available FPs that were initially advertised as being monomeric later turned out to be dimers! With any new FP you try, validate your results before making your conclusions.